A virtual microscope slide typically comprises digital data representing a magnified image of a microscope slide. Because the virtual slide is in digital form, it can be stored on a medium, such as in a computer memory, and can be transmitted over a communication network, such as the Internet, an intranet, etc., to a viewer at a remote location.
Virtual slides offer significant advantages over traditional microscope slides. In some cases, a virtual slide can enable a physician to render a diagnosis more quickly, conveniently and economically than is possible using traditional microscope slides. For example, a virtual slide may be made available to a remote user, such as a specialist in a remote location, over a communication link, enabling the physician to consult with the specialist and arrive at a more comprehensive diagnosis without the normally attendant delays associated with consultation. Alternatively, the virtual slide can be stored in digital form indefinitely, for later viewing at the convenience of the physician or specialist.
Typically, a virtual slide is generated by positioning a microscope slide (which contains a sample for which a magnified image is desired) under a microscope objective lens, capturing one or more images covering all, or a portion, of the slide, and then combining the images to create a single, integrated, digital image of the slide. It is often desirable to divide a slide into multiple regions and generate a separate image for each region. This is because, in many cases, an entire slide is often larger than the field of view of a high-power objective lens (a 20× objective, for example) and multiple images must be obtained in order to render the entire slide image at the desired 20× magnification. Additionally, the surfaces of many tissue types are uneven and contain local variations that make it difficult to capture an in-focus image of an entire slide using a fixed vertical, or z-position. As used herein, the term z-position refers the coordinate value of the z-axis of a Cartesian coordinate system. The x and y axes lie in the plane in which the stage resides. Accordingly, existing techniques typically obtain multiple images representing various regions on a slide, and combine the multiple images into an integrated image of the entire slide.
One current technique for capturing digital images of a microscopic slide is known as the start/stop acquisition method. According to this technique, multiple target points on a slide are designated for examination. A high-power objective lens (a 20× objective, for example) is positioned over the slide. At each target point, the z-position is varied and images are captured from multiple z-positions. The images are then examined to determine a desired-focus position. If one of the images obtained during the focusing operation is determined to be sufficiently in focus, it is selected as the desired-focus image for the respective target point on the slide. If none of the images is in-focus, the images are analyzed to determine a desired-focus position, the objective lens is moved to the desired-focus position, and a new image is captured. In some cases, a first sequence of images does not provide sufficient information to determine a desired focus position. In such event, it may be necessary to capture a second sequence of images within a narrowed range of z-positions before a desired-focus image is acquired. The multiple desired focus images (one for each target point) obtained in this manner may be combined to create a virtual slide.
Another approach used to generate in-focus images for developing a virtual slide includes examining the microscope slide to generate a focal map, which is an estimated focus surface created by focusing a (high-power) scanning objective lens on a limited number of points on the slide. Then, a scanning operation is performed based on the focal map. Current techniques construct focal maps by determining desired-focus information for a limited number of points on a slide. For example, such systems may select from 10 to 20 target points on a slide and use a high-power objective lens to perform a focus operation at each target point in order to determine a desired-focus position. The information obtained for those target points is then used to estimate desired-focus information for any unexamined points on the slide.
Existing start/stop acquisition systems, as described above, are relatively slow because the microscope objective lens is often required to perform multiple focus-capture operations for each designated target point on the microscopic slide. In addition, the field of view of high-power objective lenses is necessarily limited; therefore, the number of points for which desired-focus information is directly obtained may represent a relatively small portion of the entire slide.
Existing techniques for constructing focal maps also have several disadvantages. First, as described above, the use of a high-power objective lens to obtain desired-focus data for a given target point is relatively slow. Second, generating a focal map from a limited number of points on the slide can create inaccuracies in the resulting focal map. Tissue on a slide often does not have a uniform, smooth surface. Indeed, many tissue surfaces contain variations that vary across small distances (termed field-to-field variation). If a point on the surface of the tissue that has a defect or a significant local variation is selected as a target point for obtaining focus information, the local deviation can affect estimated values for desired-focus positions throughout the entire focal map. Intra-field variations can also cause focus information to be inaccurate. Even when the focus information is accurate, the mechanical nature of the microscope apparatus can cause a scan to produce an out-of-focus image due to mechanical problems; such as small motions or vibrations of the apparatus, incorrect calibration, etc. This is particularly true in the case of high-power objective lens scanning.
In yet another technique, multiple regions are defined on a microscope slide, and a focal map containing focus information for each region is generated. For each region, a plurality of z-positions are determined based on the focus information in the focal map. For each region, at least one image is captured from each of the associated z-positions. An image of each region having a desired focus quality is selected, and the selected images are combined to generate a virtual slide.